FIX variants are the source of FVIII-bypassing activity. (A-B) Neutralization of hFIX variants by mouse anti-hFIX Ab (clone HIX-1). Aliquots of hFIX-WT, hFIX-T, and hFIX-ITV or hFVIII were incubated with (white bars) or without (black bars) anti-hFIX Ab for 2 hours at 37°C. Subsequently, activities in FIX-deficient (A) or FVIII-deficient (B) plasma were determined by one-stage assay. Each bar represents the mean value of 3 independent measurements ± SEM. *P < .05, **P < .01, and ***P < .001 (Student t test) for comparison between measurements with or without Ab incubation for each protein. (C) Western blotting: lane 1 is hFIX-WT; lane 2, hFIX-T; lane 3, hFIX-ITV; lane 4, supernatant of mock-transfected 293T cells; lane 5, recombinant hFIX control (BeneFIX); and lane 6, hFIXa control. Zymogen or FIXa was detected using an anti-hFIX mAb directed toward the heavy chain of hFIX. After denaturation of the protein on SDS-PAGE, the uncleaved zymogen FIX can be identified at 70 kDa or the cleaved heavy chain at 27 kDa. Recombinant FIX (rFIX; BeneFIX) was used as a control for the FIX zymogen and FIX activated by FXIa as a control for FIXa. (D) Spurious contamination of FIX preparations by FIXa were excluded using 5 μg/mL of FIX-WT, FIX-T, FIX-ITV, or BeneFIX in an NAPTT assay sensitive to activated proteases. FIXa at a concentration of 0.2 μg/mL was used as positive control. Each bar represents the mean value of 6 independent tests with error bars ± SEM. (E) Residual FIXa activity was quantified via chromogenic assay specific for FIXa. Proteins were applied at a concentration of 2.5 μg/mL. Measured FIXa activity was negligible (no bars). (F-G) Stability of hFIX variants, hFIX-WT, or hFVIII controls in human FIX- or FVIII-deficient plasma. After incubation, samples were assayed for FIX (F) or FVIII (G) activity. Each dot represents the mean value of 3 independent tests for each sample with error bars indicating the SD.